Beta-globin-gene haplotypes, mitochondrial DNA, the Y-chromosome: their impact on the genetic epidemiology of the major structural hemoglobinopathies.

The history of the discovery of the globin beta-like globin-gene haplotypes and their importance in the understanding of hemoglobinopathies has been reviewed recently. We will add in this review more recent findings and other molecular genetic tools that can help in the understanding of the genetic epidemiology of structural hemoglobinopathies, leaving the thalassemias for a later effort.

Correction of sickle cell disease in transgenic mouse models by gene therapy.

Sickle cell disease (SCD) is caused by a single point mutation in the human betaA globin gene that results in the formation of an abnormal hemoglobin [HbS (alpha2betaS2)]. We designed a betaA globin gene variant that prevents HbS polymerization and introduced it into a lentiviral vector we optimized for transfer to hematopoietic stem cells and gene expression in the adult red blood cell lineage. Long-term expression (up to 10 months) was achieved, without preselection, in all transplanted mice with erythroid-specific accumulation of the antisickling protein in up to 52% of total hemoglobin and 99% of circulating red blood cells. In two mouse SCD models, Berkeley and SAD, inhibition of red blood cell dehydration and sickling was achieved with correction of hematological parameters, splenomegaly, and prevention of the characteristic urine concentration defect.

Ameliorating effects of fluorocarbon emulsion on sickle red blood cell-induced obstruction in an ex vivo vasculature.

In sickle cell (SS) vaso-occlusion, the culminating event is blockage of blood vessels by sickled red blood cells (SS RBCs). As shown in animal models, SS RBC-induced vaso-occlusion is often partial, allowing for a residual flow, hence oxygen delivery to partially occluded vessels could reduce vaso-occlusion. The efficacy of an oxygenated perflubron-based fluorocarbon emulsion (PFE) was tested for its anti-vaso-occlusive effects in the ex vivo mesocecum vasculature of the rat. Microvascular obstruction was induced by the infusion of deoxygenated SS RBCs into ex vivo preparations with or without pretreatment with platelet-activating factor (PAF). PAF induced enhanced SS RBC-endothelium interactions, leading to greater vaso-occlusion. Microvascular blockage resulted in increased peripheral resistance units (PRU). Deoxygenated SS RBCs caused a persistent 1.5-fold PRU increase in untreated preparations and approximately a 2-fold PRU increase in PAF-treated preparations. The greater PRU in PAF-treated preparations was caused by widespread adhesion and postcapillary blockage. Oxygenated PFE, but not deoxygenated PFE, resulted in PRU decreases to baseline values in both groups of experiments (with or without PAF). The PRU decrease caused by oxygenated PFE infusion was caused by unsickling of SS RBCs in partially occluded vessels, with no antiadhesive effect on already adherent SS RBCs as assessed by intravital microscopy. PFE had no effect on vascular tone. The efficacy of PFE appears to result from its greater capacity to dissolve oxygen (10-fold higher than plasma). The dislodgement of trapped SS RBCs and an increase in wall shear rates will help reverse the partial obstruction. Thus, oxygenated PFE is capable of reducing SS RBC-induced vaso-occlusion, and further development of this approach is advisable.

K:Cl cotransport activity is inhibited by HCO3- in knockout mouse red cells expressing human HbC.

K:Cl cotransport (KCl) was examined in transgenic mice expressing exclusively human hemoglobin C. In contrast to previous studies in early transgenic mice expressing human alpha and beta(S) and residual mouse globins, we found significant volume and pH stimulation and sensitivity to. Exposure to physiological levels of also blocked a significant fraction of KCl cotransport.

Inhibition of TNF-alpha-induced sickle RBC retention in retina by a VLA-4 antagonist.

PURPOSE: Patients with sickle cell disease have elevated circulating levels of cytokines including tumor necrosis factor (TNF) alpha. TNF-alpha stimulates expression by endothelial cells of adhesion molecules, including vascular cell adhesion molecule (VCAM) 1. Others have demonstrated that VLA-4 (alpha(4)beta(1)), a ligand for VCAM-1 or fibronectin, is present on a fraction of sickle reticulocytes. The intent of this study was to determine, using a rat model, if TNF-alpha increases retention of sickle erythrocytes in retina and if that retention can be inhibited. METHODS: TNF-alpha was given intraperitoneally to rats 5 hours before IV administration of FITC-labeled, density-separated sickle erythrocytes. After 5 minutes, rats were exsanguinated, and retinas were excised and incubated for ADPase activity, permitting the determination of the number and location of retained cells. RESULTS: TNF-alpha caused a three- to fourfold increase in retention of sickle erythrocytes in retinal capillaries (P < 0.05) but not of normal human erythrocytes. Preincubation of sickle erythrocytes with TBC772, a peptide that blocks the binding of alpha(4)beta(1) and alpha(4)beta(7), or a monoclonal antibody against VLA-4 (19H8), significantly inhibited the TNF-alpha-induced retention (P < or = 0.02), whereas a control cyclic peptide and antibody had no effect. IV TBC772 also inhibited sickle erythrocyte retention (P = 0.01). Two intravenously administered anti-fibronectin antibodies inhibited sickle cell retention as well, but an anti-rat VCAM-1 antibody did not inhibit retention. CONCLUSIONS: The authors conclude that TNF-alpha stimulates retention of sickle erythrocytes in the retinal vasculature. This increased retention can be blocked by a VLA-4 antagonist, suggesting that the cells retained after cytokine stimulation are reticulocytes. The counter-receptor for VLA-4 in this rat retina model appears to be fibronectin and not VCAM-1, based on data obtained using antibodies against these molecules.

Pleiotropic and epistatic effects in sickle cell anemia.

Sickle cell anemia is the first monogenic disease ever described, and it became the paradigm for a disease traceable to a single mutation in a single gene. Pauling's concept of "molecular disease," based on this discovery, opened a new chapter in the history of medicine. Nevertheless, at the phenotypic level, sickle cell anemia is not a monogenic disease; it is a multigenic disease. The latter is the product of pleiotropic genes (involved in secondary pathophysiologic events) and epistatic genes (same gene but with significant pathophysiologic consequences among individual=polymorphism). These secondary events are an important part of the phenotype and explain the intense interindividual differences in the severity of the disease, in spite of all the patients having the same sickle globin gene in the homozygote form. In the last decade a number of epistatic genes and pleiotropic genes have been defined, and many others are potential candidates. CHIP technology and high-throughput sequencing promise to accelerate our full multigenic understanding of this disease, contributing to a more individualized concept of disease in conjunction as we enter the new millennium.

Second generation knockout sickle mice: the effect of HbF.

Sickle transgenic mice expressing exclusively human globins are desirable for studying pathophysiology and testing gene therapy strategies, but they must have significant pathology and show evidence of amelioration by antisickling hemoglobins. Mice were generated that expressed exclusively human sickle hemoglobin with 3 levels of HbF using their previously described sickle constructs (cointegrated human miniLCRalpha2 and miniLCRbeta(S) [PNAS 89:12150, 1992]), mouse alpha- and beta-globin-knockouts, and 3 different human gamma-transgenes. It was found that, at all 3 levels of HbF expression, these mice have balanced chain synthesis, nearly normal mean corpuscular hemoglobin, and, in some cases, F cells. Mice with the least adult HbF expression were the most severe. Progressive increase in HbF from less than 3% to 20% to 40% correlated with progressive increase in hematocrit (22% to 34% to 40%) and progressive decrease in reticulocyte count (from 60% to 30% to 13%). Urine concentrating ability was normalized at high HbF, and tissue damage detected by histopathology and organ weight were ameliorated by increased HbF. The gamma-transgene that produces intermediate levels of HbF was introduced into knockout sickle mice described by Pàszty and coworkers that express the miniLCRalpha1(G)gamma(A)gammadeltabeta(S) transgene and have fetal but not adult expression of HbF. It was found that the level of HbF required to ameliorate low hematocrit and normalize urine concentrating defect was different for the miniLCRalpha2beta(S) and miniLCRalpha1(G)gamma(A)gammadeltabeta(S) mice. We conclude that knockout mice with the miniLCRalpha2beta(S) transgene and postnatal expression of HbF have sufficiently faithful sickle pathology to serve as a platform for testing antisickling interventions.

Role of epistatic (modifier) genes in the modulation of the phenotypic diversity of sickle cell anemia.

Sickle hemoglobin is the product of one mutated gene, but the disease phenotype is the product of many genes. Polymorphism among the genes responsible for the pleotropic effects can be epistatic (or modifier) genes contributing to interindividual variation that characterizes sickle cell anemia patients. Modulation in the hemoglobin F levels is associated with the beta-globin gene cluster haplotypes and to gender and chromosomal sites different from chromosome 11 influencing the severity of the disease. Coexistence of alpha thalassemia with sickle cell disease produces hematologic and clinical consequences that are beneficial in some complications but deleterious in others. There is little if any modulation of the phenotype of sickle cell anemia by coexistence of G6PD deficiency. Mutations that favor blood coagulation or thrombosis may influence the phenotype of the disease. Improved understanding of the influence of genes involved in modulating the complex pathophysiology of sickle cell disease may allow prediction of the phenotype of sickle cell patients and aid in management decisions.

Differential pathways in oxy and deoxy HbC aggregation/crystallization.

CC individuals, homozygous for the expression of beta(C)-globin, and SC individuals expressing both beta(S) and beta(C)-globins, are known to form intraerythrocytic oxy hemoglobin tetragonal crystals with pathophysiologies specific to the phenotype. To date, the question remains as to why HbC forms in vivo crystals in the oxy state and not in the deoxy state. Our first approach is to study HbC crystallization in vitro, under non-physiological conditions. We present here a comparison of deoxy and oxy HbC crystal formation induced under conditions of concentrated phosphate buffer (2g% Hb, 1. 8M potassium phosphate buffer) and viewed by differential interference contrast microscopy. Oxy HbC formed isotropic amorphous aggregates with subsequent tetragonal crystal formation. Also observed, but less numerous, were twisted, macro-ribbons that appeared to evolve into crystals. Deoxy HbC also formed aggregates and twisted macro-ribbon forms similar to those seen in the oxy liganded state. However, in contrast to oxy HbC, deoxy HbC favored the formation of a greater morphologic variety of aggregates including polymeric unbranched fibers in radial arrays with dense centers, with infrequent crystal formation in close spatial relation to both the radial arrays and macroribbons. Unlike the oxy (R-state) tetragonal crystal, deoxy HbC formed flat, hexagonal crystals. These results suggest: (1) the Lys substitution at beta6 evokes a crystallization process dependent upon ligand state conformation [i. e., the R (oxy) or T (deoxy) allosteric conformation]; and (2) the oxy ligand state is thermodynamically driven to a limited number of aggregation pathways with a high propensity to form the tetragonal crystal structure. This is in contrast to the deoxy form of HbC that energetically equally favors multiple pathways of aggregation, not all of which might culminate in crystal formation.

Primary structure of Noetia ponderosa hemoglobins: functional correlates.

Homo- and heterodimeric hemoglobins have been isolated from the red cells of the arcid clam Noetia ponderosa (Np). These hemoglobins bind oxygen cooperatively. An extensively studied dimeric hemoglobin from another arcid clam, Scapaharca inaequivalvis, exhibits a molecular mechanism for cooperative ligand binding that is radically different from tetrameric vertebrate hemoglobins. In this study, the two chains found in both Noetia hemoglobins are sequenced and compared to the hemoglobins of the related clam S. inaequivalvis to determine whether Noetia hemoglobins have the structural basis for the same unusual mechanism for cooperative ligand binding and to inquire about the structural basis of absence of tetramers. Although the Noetia sequences are homologous to the Scapharca sequences, critical differences exist. The lack of tetramerization of Np subunits is most likely related to the absence of critical residues in the A and G helices that stabilize the interdimer contact seen in the Scapharca Hb tetramer. The lower affinity of the homodimer (Np-I), but particularly the heterodimer (Np-II) with respect to the homodimer and heterotetramer of Scapharca, can be due to (i) changes in the proximal heme environment and (ii) changes in the dimer interface. Interactions between Asn 100 and the heme of the other subunit are altered in Np-II due to the substitution of this residue by methionine, possibly causing the reduced O(2) affinity of the heterodimer of Noetia. (iii) Sequence changes in the E and F helices present in Np-I and Np-II could also contribute to the effect through interfacial changes. In particular, the substitution of Val for Thr in position 72 is expected to have a substantial influence on the interface. We conclude that Np dimers have the structural basis for a direct heme-heme interaction mechanism for cooperativity, as in Scapharca, but there are enough sequence changes to suggest that the pathway of interaction might be somewhat different.

Primitive haematopoietic progenitors in the blood of patients with sickle cell disease appear to be endogenously mobilized.

To investigate whether haematopoietic stem cells in patients with sickle cell (SS) disease might be altered, we examined the number and cycling status of 5-week long-term culture-initiating cells (LTC-ICs) and in vitro multilineage colony-forming cells (CFCs) present in the blood of a large and clinically diverse group of SS patients. The concentrations of both of these cell types per ml of blood varied over a wide range in individual patients, but, on average, were significantly elevated above normal values ( approximately sevenfold and 15-fold respectively) and to an even greater extent than the lineage-restricted CFCs in the same samples. Wide variations in the concentration of circulating progenitors, particularly the LTC-ICs, were also seen over time (in concert with changes in the white blood cell count) in SS patients. [3H]-Thymidine suicide assays showed most of the CFCs and LTC-ICs in SS blood to be quiescent like their counterparts in normal blood. However, by comparison with historical data, the SS progenitors could be recruited into the cycle more quickly (i.e. within 2 vs. 3 d), thus showing the same kinetics of activation exhibited by 'mobilized' progenitors from patients given chemotherapy and exogenous growth factors. Taken together, these findings implicate previously documented increases in endogenous Steel factor, interleukin 3 and granulocyte-macrophage colony-stimulating factor levels in SS patients in the establishment of a chronically mobilized progenitor phenotype.

Velocity measurements of normal and sickle red blood cells in the rat retinal and choroidal vasculatures.

The purpose of this study was to develop an in vivo, noninvasive method to assess the velocities of normal and sickle red blood cells (RBCs) in the retinal and choroidal vasculatures of rats. Human and rat RBCs were isolated from whole blood, labeled with fluorescein isothiocyanate (FITC), and administered intravenously to anesthetized rats. A Rodenstock scanning laser ophthalmoscope (SLO) was used to image the FITC-labeled RBCs as an NTSC video signal. Video sequences of RBC transit in the retinal (pigmented rats) and choroidal (albino rats) vessels were captured directly to digital format. Following in vivo angiography, the animals were sacrificed, the eyes enucleated, and retinas prepared by our adenosine diphosphatase vascular labeling technique for viewing by conventional optical microscopy. Although rat and normal human RBCs differ slightly in size, their velocities were similar in the retinal arteries and capillaries (within 4%). Velocities of RBCs from sickle cell patients (sRBCs) were slower by 12 and 9% in arteries and by 38 and 25% in capillaries, compared to rat and normal human RBCs, respectively. Compared to velocities in retinal capillaries, the velocities in choroidal capillaries were much slower for rat RBCs (77%), normal human RBCs (79%), and sRBCs (67%). In contrast to normal human RBCs, sRBCs were often retained transiently in retinal capillaries at preferred sites, but in choroidal capillaries large numbers of cells were retained for extended periods. SLO imaging of FITC-labeled RBCs in rat retina and choroid provided a reliable method for evaluating normal and abnormal hemodynamics.

Hemoglobin C in transgenic mice: effect of HbC expression from founders to full mouse globin knockouts.

When present in the homozygous form, hemoglobin C (HbC, CC disease) increases red cell density, a feature that is the major factor underlying the pathology in patients with SC disease (Fabry et al., JCI 70, 1315, 1982). The basis for the increased red cell density has not yet been fully defined. We have generated a HbC mouse in which the most successful founder expresses 56% human alpha and 34% human beta(C). We introduced knockouts (KO) of mouse alpha- and beta-globins in various combinations. In contrast to many KO mice, all partial KOs have normal MCH. Full KOs that express exclusively HbC and no mouse globins have minimally reduced MCH (13. 7 +/- 0.3 pg/cell vs 14.5 +/- 1.0 for C57BL/6) and a ratio of beta- to alpha-globin chains of 0.88 determined by chain synthesis; hence, these mice are not thalassemic. Mice with beta(C) > 30% have increased MCHC, dense reticulocytes, and increased K:Cl cotransport. Red cell morphology studied by SEM is strikingly similar to that of human CC cells with bizarre folded cells. We conclude that red cells of these mice have many properties that closely parallel the pathology of human disease in which HbC is the major determinant of pathogenesis. These studies also establish the existence of the interactions with other gene products that are necessary for pleiotropic effects (red cell dehydration, elevated K:Cl cotransport, morphological changes) that are also present in these transgenic mice, validating their usefulness in the analysis of pathophysiological events induced by HbC in red cells.

Megalophallus as a sequela of priapism in sickle cell anemia: use of blood oxygen level-dependent magnetic resonance imaging.

Priapism is a common complication of sickle cell anemia. We report a little known sequela of priapism: painless megalophallus, with significant penile enlargement. The patient had had an intense episode of priapism 9 years previously and his penis remained enlarged. Blood oxygen level-dependent magnetic resonance imaging revealed enlarged, hypoxic corpora cavernosa. Megalophallus probably resulted from permanent loss of elasticity of the tunica albuginea due to severe engorgement during the episode of priapism. This sequela needs to be recognized by physicians because no intervention is necessary and sexual function seems to remain intact.